TW201908493A - DNA polymerase activity enhancing PCR buffer composition with increased gene mutation specificity - Google Patents

Abstract

The present invention relates to a DNA polymerase having increased gene mutation specificity and a PCR buffer composition for increasing activity of the DNA polymerase. More specifically, provided, in the present invention, are a DNA polymerase in which a mutation is induced at a specific amino acid position to increase gene mutation specificity, a nucleic acid sequence encoding the polymerase, a vector comprising the nucleic acid sequence, and a host cell transformed with the vector. In addition, provided are a method for in vitro detecting one or more gene mutations or SNPs in one or more templates by using a DNA polymerase having increased gene mutation specificity, a composition for detecting a gene mutation or SNP comprising the DNA polymerase.

Description

   PCR buffer composition for enhancing DNA polymerase activity with increased specificity of gene mutation   

The present invention relates to a PCR buffer composition for enhancing DNA polymerase activity enhancement with increased specificity of genetic mutations, and in particular to a PCR buffer composition for enhancing the activity of DNA polymerases, and in particular to a DNA polymerase activity that induces mutations at specific amino acid positions to increase specificity of genetic mutations. Enhancement PCR buffer composition, PCR kit for detecting gene mutation or SNP comprising said PCR buffer composition and / or DNA polymerase with increased specificity of gene mutation, and using said kit at least one gene or a mutation detection method for detecting SNP from more than one template in vitro (in vitro).

Since the first human genome sequence was discovered, researchers' research has focused on exploring genetic differences among individuals such as single nucleotide mutations (Single Nucleotide Polymorphisms, SNPs). Single nucleotide mutations in the genome are associated with different drug resistance or susceptible constitutions of various diseases, which has become more and more clear, so it has become the main concern. Knowledge of future medical-related nucleotide variants can be applied to treatments that are genetically supplied by individuals and can prevent ineffective or side-effect drug treatments (Shi, Expert Rev. Mol. Diagn. 1, 363-365 (2001)). The development of efficient nucleotide variation identification technology in time and cost will bring further development of pharmacogenetics.

SNPs occupy the major genetic variation in the human genome and can induce more than 90% of individual differences. (Kwok, Annu. Rev. Genomics Hum, Genet. 2, 235-258 (2001); Kwok and Chen, Curr. Issues Mol. Biol. 5, 43-60 (2003); Twyman and Primrose, Pharmacogenomics 4, 67- 79 (2003)). To detect these genetic variations and other nucleic acid variations such as mutations, various methods can be used. For example, identification of a target nucleic acid variant can be achieved by hybridizing a nucleic acid sample to be analyzed with a hybridization primer specific for a sequence variant under appropriate hybridization conditions (Guo et al., Nat. Biotechnol. 15, 331-335 (1997)).

However, it was found that this hybridization method cannot meet the clinical needs, especially in terms of the sensitivity necessary for the measurement. Therefore, PCR has been widely used in diagnostic methods for the detection of mutations such as molecular biology, SNPs, and other allelic sequence variants (Saiki et al., Science 239, 487-490 (1988)). In the presence of the target, the target nucleic acid to be detected is amplified by polymerase chain reaction (PCR) before hybridization. Hybridization primers for such assays typically use single-stranded oligonucleotides. Specific examples of modifications of the assay include the use of fluorescent hybridization probes (Livak, Genet. 73-5 2017-07-12 Anal. 14, 143-149 (1999)). In general, attempts have been made to automate the determination of SNP and other sequence variations (Gut, Hum. Mutat. 17, 475-492 (2001)).

Countermeasure lines for sequence mutation-specific hybridization known in the art are provided by so-called gene mutation-specific amplification. In this detection method, a mutation-specific amplification primer has been used in the amplification process. Generally, the 3'-end of the primer has a so-called differential terminal nucleotide residue, and the residue is only one target nucleic acid to be detected. Specific mutations are complementary. In this method, nucleotide variants are measured in the presence or absence of a DNA product after PCR amplification. The principle of gene mutation-specific amplification is based on the formation of a canonical or atypical primer-template complex at the end of a gene mutation-specific amplification primer. At the ends of the precisely matched 3 'primers, amplification by DNA polymerase is generated, while at the ends of the mismatched primers, extension is suppressed.

For example, U.S. Patent No. 5,595,890 discloses a gene mutation-specific amplification method and application thereof, such as a method for detecting clinically relevant point mutations in a k-ras tumor gene. In addition, US Patent No. 5,521,301 discloses an allele-specific amplification method for genotyping of the ABO blood group system. In contrast, U.S. Patent No. 5,639,611 discloses a method of using allele-specific amplification related to detecting a point mutation that causes sickle type anemia. However, mutation-specific or allele-specific amplification has a problem of low selectivity, and therefore requires more complicated, time- and cost-intensive optimization steps.

The method for detecting sequence mutations, polymorphisms, and point mutations are particularly required when the sequence variation to be detected is insufficient compared with the dominant mutation of the same nucleic acid fragment (or the same gene). Amplification (or mutation-specific amplification).

This occurs, for example, when sporadic tumor cells are detected in a bodily fluid such as blood, serum, or plasma by genetic mutation-specific amplification (US Patent No. 5,496,699). To this end, DNA is first isolated from body fluids such as blood, serum, or plasma, while DNA consists of insufficient sporadic tumor cells and excess non-proliferating cells. Therefore, in the presence of excess wild-type DNA, important mutations in tumor DNA in the k-ras gene should be detected from multiple replications.

All methods disclosed in the prior art for the specific amplification of gene mutations, although using 3'-differential nucleotide residues, even if the target nucleic acid does not completely match the sequence variant to be detected, In the presence of DNA polymerase, it has the disadvantage of lower level of primer extension. In particular, when a particular sequence variant is detected by containing an excess of background nucleic acid from another sequence variant, a false positive result can result. Another disadvantage of the known methods is the need to use 3'-terminal differential oligonucleotide residues. The disadvantage of this PCR-based method is mainly due to the incompatibility of the polymerase used in the method for sufficiently distinguishing mismatched bases. Therefore, it is not yet possible to directly obtain clear information about the presence or absence of mutations by PCR. To date, additional time and cost-intensive purification and assay methods are needed to unambiguously diagnose mutations. Therefore, new methods to increase the selectivity of gene mutation-specific or allele-specific PCR amplification will greatly affect the reliability and robustness of direct gene mutation or SNP analysis by PCR.

Therefore, there is a need to continue to study DNA polymerases with increased specificity for genetic mutations and optimal reaction buffers that mix the various substances that allow the DNA polymerase to perform its function.

The present inventors have devoted to the development of a novel DNA polymerase capable of improving gene mutation-specific PCR amplification selectivity and a reaction buffer for improving its activity, and the results have confirmed that the induction of amino acid residues at specific positions of Taq polymerase During mutation, gene mutation specificity increased significantly. When controlling the concentration of KCl, (NH ₄ ) ₂ SO ₄ and / or TMAC (Tetra methyl ammonium chloride) in PCR buffer composition, The activity of a DNA polymerase having an increased specificity of a genetic mutation is enhanced, thereby completing the present invention.

For this reason, an object of the present invention is to provide a PCR buffer composition for enhancing DNA polymerase activity with increased specificity for gene mutation.

Another object of the present invention is to provide a PCR kit for detecting a gene mutation or SNP, which comprises the PCR buffer composition.

Another object of the present invention is to provide a detection method for detecting at least one gene mutation or SNP in vitro from more than one template using the PCR kit.

In order to achieve the object, the present invention provides a PCR buffer composition for enhancing DNA polymerase activity with increased specificity of gene mutation, wherein the composition comprises 25 to 100 mM KCl; 1 to 15 mM (NH ₄ ) ₂ SO ₄ ; final pH is 8.0 to 9.0.

In an embodiment of the present invention, the KCl concentration is 60 to 90 mM.

In yet another embodiment of the present invention, the (NH ₄ ) ₂ SO ₄ concentration is 2.5 to 8 mM.

In another embodiment of the present invention, the KCl concentration is 70 to 80 mM, and the (NH ₄ ) ₂ SO ₄ concentration is 4 to 6 mM.

The present invention provides a PCR buffer composition for enhancing DNA polymerase activity with increased specificity for genetic mutations, wherein the PCR buffer composition further comprises 5 to 80 mM TMAC (Tetra methyl ammonium chloride) .

In one embodiment of the present invention, the KCl concentration is 40 to 90 mM.

In another embodiment of the present invention, the (NH ₄ ) ₂ SO ₄ concentration is 1 to 7 mM.

In another embodiment of the present invention, the TMAC (Tetra methyl ammonium chloride) concentration is 15 to 70 mM, the KCl concentration is 50 to 80 mM, and the (NH ₄ ) ₂ SO ₄ concentration It is 1.5 to 6 mM.

As in another embodiment of the present invention, wherein the PCR buffer composition further comprises Tris. Cl and MgCl ₂ .

The invention provides a PCR kit for detecting a gene mutation or SNP, which comprises the PCR buffer composition.

In yet another embodiment of the present invention, the PCR kit may further include a DNA polymerase of Taq polymerase consisting of the amino acid sequence of SEQ ID NO: 1 (the base sequence of SEQ ID NO: 6), The DNA polymerase may include (a) the substitution of the 507th amino acid residue in the amino acid sequence of SEQ ID NO: 1; and (b) the 536th amino acid sequence in the amino acid sequence of SEQ ID NO: 1 Substitution of amino acid residues, substitution of the 660th amino acid residue, or substitution of the 536th and 660th two amino acid residues.

In another embodiment of the present invention, wherein the substitution of the 507th amino acid residue is glutamic acid (E) by lysine (K), and the substitution of the 536th amino acid residue Is that arginine (R) is replaced by lysine (K), and the substitution of the 660th amino acid residue is that arginine (R) is replaced by valine (V).

In yet another embodiment of the present invention, the PCR kit further comprises: (i) nucleoside triphosphate; (ii) a quantitative reagent for binding to double-stranded DNA; (iii) a polymerase blocking antibody; (iv) One or more control values or sequences; (v) one or more templates.

The invention also provides a detection method for detecting at least one gene mutation or SNP in vitro from more than one template by using the PCR kit.

Compared with the prior art, the present invention has the following beneficial effects: The present invention provides an optimal PCR buffer composition which can make a DNA polymerase with increased specificity of genetic mutations perform its function, and is specific to having an increased genetic mutation The use of sexual DNA polymerase together can significantly increase the activity of the DNA polymerase, and can achieve reliable gene mutation-specific amplification. In addition, the kit containing the PCR buffer composition of the present invention and / or a DNA polymerase with increased gene specificity can effectively detect gene mutations or SNPs, and thus can be effectively used for medical diagnosis of diseases and recombinant DNA the study.

The following drawings describe the non-limiting embodiments in detail to make other features, objects, and advantages of the present invention more obvious:

Figure 1 is a description of the preparation process of Taq DNA polymerase containing R536K, R660V, and R536K / R660V mutations, respectively, where (a) is a schematic diagram of fragment PCR and overlapping PCR, and (b) shows the electrophoresis of amplified products in fragment PCR The results were confirmed, and (c) shows the results of confirming the amplified products by electrophoresis after amplifying the entire length by overlapping PCR.

Fig. 2 shows the results of electrophoresis confirmation of the pUC19 vector, which was SAP-treated with the restriction enzyme EcoRI / XbaI for the extraction of the gel, and the purified overlapping PCR product of Fig. 1 (c).

Figure 3 is a schematic diagram of fragment PCR and overlapping PCR during the preparation of Taq DNA polymerase containing E507K, E507K / R536K, E507K / R660V, and E507K / R536K / R660V mutations, respectively.

Fig. 4 shows the results of electrophoresis confirmation of the pUC19 vector which was subjected to SAP treatment after being decomposed with the restriction enzyme EcoRI / XbaI for the extraction of the gel and the purified overlapping PCR product of Fig.

FIG. 5 is a schematic diagram showing a process of preparing a PCR template by collecting oral epithelial cells.

FIG. 6 shows the results of AS-qPCR on rs1408799 using E507K / R536K, E507K / R660V and E507K / R536K / R660V Taq polymerases of the present invention; as a control group, Taq polymerase including E507K mutation was used.

FIG. 7 shows the results of AS-qPCR on rs1015362 using the E507K / R536K, E507K / R660V, and E507K / R536K / R660V Taq polymerases of the present invention; as a control group, Taq polymerase including E507K mutation was used.

FIG. 8 shows the results of AS-qPCR on rs4911414 using E507K / R536K, E507K / R660V, and E507K / R536K / R660V Taq polymerases of the present invention; as a control group, Taq polymerase including E507K mutation was used.

FIG. 9 is a graph showing confirmation of the delay effect of mismatch amplification based on changes in KCl concentration in a reaction buffer using E507K, E507K / R536K, E507K / R660V, and E507K / R536K / R660V Taq polymerases of the present invention.

FIG. 10 shows the results of confirming PCR products by electrophoresis after determining the optimal KCl concentration in the reaction buffer by fixing the concentration of (NH ₄ ) ₂ SO ₄ and changing the KCl concentration for amplification.

FIG. 11 shows the results of confirming PCR products by electrophoresis after determining the optimal (NH ₄ ) ₂ SO ₄ concentration in the reaction buffer by fixing KCl concentration and changing the (NH ₄ ) ₂ SO ₄ concentration for amplification.

FIG. 12 is a graph showing a delay effect of confirming mismatch amplification based on a change in (NH ₄ ) ₂ SO ₄ concentration in a reaction buffer.

FIG. 13 is a graph showing the concentration of KCl and (NH ₄ ) ₂ SO ₄ fixed in the reaction buffer, and the delay effect of mismatch amplification was confirmed based on the change in TMAC concentration.

FIG. 14 is a graph showing the delay effect of mismatched amplification by confirming the concentration of TMAC and (NH ₄ ) ₂ SO ₄ in the reaction buffer and changing the KCl concentration.

The present invention will be described in detail below.

As described above, in order to improve the shortcomings of the gene mutation-specific amplification method disclosed in the prior art, it is necessary to continuously develop a DNA polymerase having an increased specificity of the gene mutation and a mixture of a plurality of types of DNA polymerases capable of exerting their functions. The optimal reaction buffer for the substance, and the development of these methods will greatly affect the reliability and robustness of direct genetic mutation or SNP analysis by PCR. The present inventors have devoted to the development of a novel DNA polymerase capable of improving gene mutation-specific PCR amplification selectivity and a reaction buffer for improving its activity, and the results confirmed that the induction of amino acid residues at specific positions of Taq polymerase was confirmed During mutation, gene mutation specificity increased significantly. When the concentration of KCl, (NH ₄ ) ₂ SO ₄ and / or TMAC (Tetra methyl ammonium chloride) in the PCR buffer composition was controlled, The activity of the DNA polymerase having increased specificity of the mutation of the gene is enhanced, thereby completing the present invention.

The present invention provides an optimal PCR buffer composition that allows a DNA polymerase with increased genetic mutation specificity to perform its function. When used together with a DNA polymerase with increased genetic mutation specificity, the DNA polymerase is significantly improved. Activity for reliable mutation-specific amplification. In addition, the kit containing the PCR buffer composition of the present invention and / or a DNA polymerase with increased gene specificity can effectively detect gene mutations or SNPs, and thus can be effectively used for medical diagnosis of diseases and recombinant DNA the study.

The terms used herein will be described below.

As used herein, "amino acid" refers to any monomeric unit that can be introduced into a peptide, polypeptide, or protein. As used herein, the term "amino acid" includes the following 20 naturally or genetically encoded alpha -amino acids: alanine (Ala or A), spermine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G) , Histidine (His or H), Isoleucine (Ile or I), Leucine (Leu or L), Lysine (Lys or K), Methionine (Met or M), Phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y) , Valine acid (Val or V).

Amino acids are generally organic acids and may include substituted or unsubstituted amine groups, substituted or unsubstituted carboxyl groups, and at least one side chain or group, or any analogues of these groups . Exemplary side chains include mercapto, seleno, sulfonyl, alkyl, aryl, fluorenyl, keto, azide, hydroxyl, hydrazino, cyano, halo, hydrazine, alkenyl, alkynyl , Ether, borate, borate, dioxo, phosphinosulfonate, phosphine, heterocycle, ketene, imine, aldehyde, ester, thioacid, hydroxylamine or any of these groups combination.

Other exemplary amino acids include, but are not limited to, amino acids containing photoactivatable cross-linking agents, metal-bound amino acids, spin-labeled amino acids, fluorescent amino acids, metal-containing amino acids, having Novel functional amino acids, amino acids covalently or non-covalently interacting with other molecules, photocagetable and / or photoisomerizable amino acids, radioactive amino acids, containing biotin or biotin Analogous amino acids, glycosylated amino acids, other carbohydrate modified amino acids, amino acids containing polyethylene glycol or polyether, heavy atom substituted amino acids, chemically decomposable and / or Photodegradable amino acids, carbon-linked sugar-containing amino acids, redox-active amino acids, sulfur-containing carboxylic acids, and amino acids containing at least one toxic moiety.

In the DNA polymerase of the present invention, the term "mutant" refers to a recombinant polypeptide comprising one or more amino acid substitutions relative to a naturally occurring or unmodified DNA polymerase.

The term "thermostable polymerase" (refers to a thermostable enzyme) refers to having sufficient thermal resistance, maintaining sufficient activity to achieve subsequent polynucleotide extension reactions, and the time course required to achieve double-stranded nucleic acid denaturation It does not undergo irreversible denaturation (deactivation) during medium temperature treatment. As used herein, it applies to cycling temperatures in reactions such as PCR. The irreversibility here refers to the complete loss of permanent enzyme activity. For thermostable polymerase, enzyme activity refers to catalyzing a combination of nucleotides in a suitable manner to form complementary polynucleotide extension products on a template nucleic acid strand. Thermostable DNA polymerases derived from thermophilic bacteria include, for example: from Thermatoga maritime , Thermus acquaticus , Thermus thermophilus , Thermusflavus ), Thermus filiformus , Thermus filiformus Sps17, Thermus thermophilus Z05 , Thermus caldophilus , Bacillus caldotenax , New Apollo ( Thermotoga neapolitana ), DNA polymerase of Thermosipho africanus .

The term "thermal activity" refers to an enzyme that retains its catalytic performance at temperatures (ie, 45-80 ° C) commonly used in the reverse transcription or adhesion / extension stages of RT-PCR and / or PCR reactions. Thermostable enzymes are enzymes that do not irreversibly inactivate or denature when treated with the high temperature required for denaturation of nucleic acids. And thermoactive enzymes may or may not be thermostable. Thermoactive DNA polymerase may include, but is not limited to, DNA or RNA that is dependent on a thermophilic or mesophilic species.

The term "host cell" refers to unicellular prokaryotes and eukaryotic organisms (such as bacteria, yeast, and actinomycetes) and single cells from higher plants or animals for use in culture in cell culture media.

The term "vector" is a DNA molecule such as a plastid, a phage, an artificial chromosome, or the like that can be replicated and can transfer genes or the like to foreign DNA to a recipient cell. "Plastid", "carrier" or "plasmid carrier" are used interchangeably herein.

The term "nucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in the form of a single strand or double strand, and unless It is specifically stated that analogs of natural nucleotides may be included.

The term "nucleic acid" or "polynucleotide" refers to a polymer of DNA or RNA polymer, or an analogue thereof. Nucleic acids can be, for example, chromosomes or chromosome fragments, vectors (e.g., expression vectors), expression cassettes, naked DNA or RNA polymers, polymerase chain reaction (PCR) products, oligonucleotides, probes or primers, or contain Mentioned matter. Nucleic acids can be, for example, single-stranded, double-stranded, or triple-stranded, but are not limited to any particular length. Unless otherwise specified, a particular nucleic acid sequence includes or can encode a complementary sequence in addition to any sequence specified.

The term "primer" refers to a polynucleotide that can serve as a starting point for nucleic acid synthesis in the direction of the template when the polynucleotide is under extended initiation conditions. Primers can also be used in a variety of other oligonucleotide-mediated synthetic processes, including de novo synthesis of RNA and as promoters for in vitro transcription-related processes. Primers are usually single-stranded oligonucleotides (eg, oligodeoxyribonucleotides). The proper length of a primer is usually in the range of 6 to 40 nucleotides, more typically in the range of 15 to 35 nucleotides and depends on the intended use. Short primer molecules generally require lower temperatures to form a sufficiently stable hybrid complex with the template. Primers do not need to reflect the correct sequence of the template, but the primers must be sufficiently complementary to hybridize with the template to be extended. In specific embodiments, the term "primer pair" refers to a 5 'sense primer that includes a complementary hybridization to the 5' end of a nucleic acid sequence to be amplified, and a 3'-antisense primer that hybridizes to the 3 'end of a sequence to be amplified. Group of primers. If necessary, primers can be labeled by incorporating tags that can be detected by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful markers include: 32P, fluorescent dyes, electron density reagents, enzymes (typically used in ELISA assays), biotin or haptens, and proteins that can use antisera or monoclonal antibodies.

The term "5'-nuclease (nuclease) probe" refers to an oligonucleotide comprising at least one luminescent label moiety for performing a 5'-nuclease reaction to detect a target nucleic acid. In some embodiments, for example, a 5'-nuclease probe contains only a single luminescent moiety (eg, a fluorescent dye, etc.). In certain embodiments, the 5'-nuclease probe comprises a self-complementary region such that the probe can form a hairpin structure under selected conditions. In some embodiments, the 5'-nuclease probe comprises two or more labeled moieties, one of the two tags is separated or broken down from the oligonucleotide, and the radiation intensity is increased to be released. In certain embodiments, the 5'-nuclease probe is labeled with two different fluorescent dyes, such as a 5'-terminal reporter dye and a 3'-terminal quenching dye or a partial label. In some embodiments, the 5'-nuclease probe is further labeled at the end or at one or more positions other than the end. When the probe is intact, energy transfer usually occurs between the two fluorescent materials, so that the fluorescence emission from the reporter dye is quenched more than a certain portion. During the extension step of the polymerase chain reaction, for example, a 5'-nuclease probe bound to a template nucleic acid has an activity such that the fluorescent emission of the reporter dye is no longer quenched, such as by Taq polymerase or other polymerization The 5 'to 3'-nuclease activity of the enzyme is broken down. In some embodiments, the 5'-nuclease probe can be labeled or partially labeled with two or more different reporter dyes and a 3'-terminal quenching dye.

The terms "FRET" or "fluorescence resonance energy transfer" or "forester resonance energy transfer" refer to two or more chromophores, a donor chromophore, and an acceptor chromophore (called a quencher). Between energy transfers. The donor is usually excited by emitting light of the appropriate wavelength, transferring energy to the acceptor. Receptors usually re-emit moving energy in the form of re-emitting light of different wavelengths. When the acceptor is a "dark" quencher, the moving energy is dispersed in a form other than light. Whether a particular fluorescent substance acts as a donor or acceptor depends on the nature of FRET for other members. Common donor-acceptor pairs include FAM-TAMRA pairs. Commonly used quenchers are DABCYL and TAMRA. Common dark quenchers include: BlackHole Quenchers ^TM (BHQ), (Biosearch Technologies, Inc., Novato, Cal.), Iowa Black ^TM (Integrated DNA Tech., Inc., Coralville, Iowa), BlackBerry ^TM Quencher 650 ( (BB-650) (Berry & Assoc., Dexter, Mich.).

When referring to nucleic acid bases, nucleoside triphosphates, or nucleotides, the term "conventional" or "natural" refers to the polynucleotides that occur naturally (ie, dATP, dGTP, dCTP, dTTP for DNA) ), And dITP and 7-deaza-dGTP are often used in place of dGTP, and can be used in place of dATP in DNA synthesis reactions such as sequencing.

When referring to nucleic acid bases, nucleosides, or nucleotides, the term "unconventional" or "modification" encompasses the modification, derivation of conventional bases, nucleosides, or nucleotides that naturally occur in a particular polynucleotide. Or similar. Compared to conventional dNTPs, specific unconventional nucleotides are modified at the 2 'position of ribose. Therefore, even if the naturally occurring nucleotide of RNA is a ribonucleotide (ie, ATP, GTP, CTP, UTP, collectively rNTP), the nucleotide has a hydroxyl group at the 2 'position of the sugar, and dNTP is relatively absent Therefore, as used herein, ribonucleotides are unconventional nucleotides that serve as substrates for DNA polymerases. As used herein, non-conventional nucleotides include, but are not limited to, compounds used as nucleic acid sequencing terminator. Exemplary terminator compounds include, but are not limited to, compounds having a 2 ', 3'-dideoxy structure, known as dideoxynucleoside triphosphates. Dideoxynucleoside triphosphates ddATP, ddTTP, ddCTP, and ddGTP are collectively referred to as ddNTP. Other examples of terminator compounds include 2'-PO4 analogs of ribonucleotides. Other unconventional nucleotides include, but are not limited to, thioddNTP ([[ α ] -S] dNTP), 5 '-[ α ] -borano-dNTP, [ α ] -methyl-phosphonic acid dNTP, ribose Nucleoside triphosphate (rNTP). Unconventional bases can be radioactive isotopes such as 32P, 33P or 35S; fluorescent labels; chemiluminescent labels; bioluminescent labels; hapten labels such as biotin; or enzyme labels such as streptavidin or avidin mark. Fluorescent labels can include negatively charged dyes such as those of the luciferin family, or neutrally charged dyes such as those of the rhodamine family, or positively charged dyes such as the cyanine family. Dyes of the luciferin family include, for example, FAM, HEX, TET, JOE, NAN, and ZOE. Rhodamine family dyes include Texas Red, ROX, R110, R6G and TAMRA. Various dyes or nucleotides labeled with FAM, HEX, TET, JOE, NAN, ZOE, ROX, R110, R6G, Texas Red, and TAMRA are passed through Perkin-Elmer (Boston, Massachusetts), Applied Biosystems (Fox Specialty, CA), Invitrogen / Molecular Probes (Eugene, Oregon). The cyanine family dyes include Cy2, Cy3, Cy5, and Cy7, and are sold through the GE Healthcare UK Limited (Amersham Place, Little Chalfont, Buckinghamshire, England) market.

The term "mismatch discrimination" refers to the use of a nucleic acid (such as a primer or other oligonucleotide) to extend a nucleic acid (such as a primer or other oligonucleotide) in a template-dependent manner by attaching (e.g., covalently) one or more nucleotides to a nucleic acid. The ability to distinguish biocatalytic (such as polymerase, ligase, etc.) biosynthetic sequences from mismatch-containing sequences. The term "mismatch discrimination" refers to a mismatch-contained (about complementary) sequence discrimination from a 3 'terminal nucleic acid that has a mismatch compared to a template where an extended nucleic acid (such as a primer or other oligonucleotide) hybridizes to a nucleic acid. Biocatalytic capacity of perfectly complementary sequences. In some embodiments, the extended nucleic acid comprises a mismatch at the 3 'end for a perfectly complementary sequence. In some embodiments, the extended nucleic acid comprises a mismatch at the second (N-1) 3'-position and / or N-2 position of the end for a fully complementary sequence.

Unless defined otherwise, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art.

The present invention relates to a PCR buffer composition for enhancing DNA polymerase activity with increased specificity for genetic mutations, said composition comprising 25 to 100 mM KCI; 1 to 15 mM (NH ₄ ) ₂ SO ₄ ; and a final pH of 8.0 To 9.0.

The polymerase used in PCR should use an optimal reaction buffer mixed with various substances to perform its function. The reaction buffer usually contains a factor that stabilizes pH, a metal ion serving as a cofactor, and a stabilizing component that prevents denaturation of the polymerase.

The KCl can be used as an essential factor for stabilizing the enzyme, helping the primer to pair with the target DNA. In the present invention, the optimal concentration is determined by adjusting the concentration of KCl in the reaction buffer to confirm that the amplification caused by mismatching is delayed to the maximum, while the high cation concentration of the amplification efficiency caused by matching is not reduced.

Using E507K, E507K / R536K, E507K / R660V, and E507K / R536K / R660V Taq polymerase, confirm the mismatch amplification delay effect caused by the change in KCl concentration in the reaction buffer. The results are shown in Figure 9. / R660V Taq polymerase, even if there is no KCl in the reaction buffer, its mismatch amplification delay is excellent; for E507K / R536 and E507K / R660V, at 50mM; as a control group E507K, at 100mM, mismatch amplification The delay effect is excellent. As a result, for the threshold of KCl concentration, E507K / R536K / R660V Taq polymerase is the lowest, and E507K / R536K and E507K / R660V are lower than E507K.

In order to further derive the appropriate KCI concentration, E507K / R536K / R660V Taq polymerase was used to fix the (NH ₄ ) ₂ SO ₄ concentration in the reaction buffer to a certain value, and multiple KCl concentrations were used for amplification. The results of the amplification products were confirmed by electrophoresis. As shown in FIG. 10, it was confirmed that the appropriate KCI concentration was 75 mM.

Therefore, the KCl concentration in the PCR reaction buffer composition of the present invention may be 25-100 mM, preferably 60-90 mM, more preferably 70-80 mM, and most preferably 75 mM.

When the KCl concentration is lower than 25 mM, it has no effect on the amplification of general targets, but it can reduce the difference between the amplification caused by matched primers and the amplification caused by mismatched primers. When the concentration is higher than 100 mM, the general The target has a problem of reduced amplification efficiency.

For the PCR reaction buffer composition, the (NH ₄ ) ₂ SO ₄ is an essential co-factor for enzyme activity, and is used with Tris to increase the polymerase activity. In one embodiment of the present invention, based on the above derivation results, the KCl concentration in the reaction buffer was fixed at 75 mM, and the concentration of (NH ₄ ) ₂ SO ₄ was varied from 2.5 mM to 25 mM to confirm the appropriate ( NH ₄ ) ₂ SO ₄ concentration.

As a result, as shown in FIG. 11, the amplification product was confirmed at a concentration of (NH ₄ ) ₂ SO ₄ of 2.5 mM to 15 mM, and an appropriate concentration of (NH ₄ ) ₂ SO ₄ was 5 mM.

Further, when the concentration of (NH ₄ ) ₂ SO ₄ was close to 5 mM (2.5 mM, 5 mM, and 10 mM, respectively), AS-qPCR was performed. The results are shown in FIG. 12. Although the difference in Ct value was the largest at 10 mM, It was confirmed that Ct was slightly delayed in the matching amplification, and the peak was dumped downward. Therefore, it was determined that the appropriate (NH ₄ ) ₂ SO ₄ concentration was 5 mM.

Therefore, the concentration of (NH ₄ ) ₂ SO ₄ in the PCR reaction buffer composition of the present invention may be 1-15 mM, preferably 2.5-8 mM, more preferably 4-6 mM, and most preferably 5 mM.

When the concentration of (NH ₄ ) ₂ SO ₄ is less than 1 mM, it has no effect on the amplification of general targets, but it can reduce the difference between the amplification caused by matched primers and the amplification caused by mismatched primers. When the concentration is high At 15 mM, there is a problem that the amplification efficiency of a general target decreases.

Therefore, the optimal PCR buffer composition of the present invention may contain 70 to 80 mM KCl; 4 to 6 mM (NH ₄ ) ₂ SO ₄ ; and a final pH of 8.0 to 9.0.

The PCR buffer composition of the present invention may further include 5 to 80 mM TMAC (Tetra methyl ammonium chloride).

TMAC is commonly used to reduce amplification caused by mismatches or improve stringency of hybridization reactions. In one embodiment of the present invention, based on the above derivation results, the KCl concentration in the reaction buffer is fixed at 75 mM, the concentration of (NH ₄ ) ₂ SO ₄ is fixed at 5 mM, and the TMAC concentration is varied from 0 mM to 80 mM. To confirm the appropriate TMAC concentration.

The results are shown in FIG. 13. The appropriate TMAC concentration was confirmed to be 70 mM for E507K / R536K Taq polymerase and 25 mM for E507K / R536K / R660V Taq polymerase. The TMAC concentration was further fixed at 25 mM, the concentration of (NH ₄ ) ₂ SO ₄ was fixed at 2.5 mM, and the KCl concentration was adjusted to 20, 40, 60, and 80 mM, respectively. rs1015362 and rs4911414, the appropriate KCl concentration is 60mM.

When the TMAC concentration exceeds 80 mM, the amplification efficiency decreases, so it is preferable to use TMAC within the above range.

Therefore, when the PCR buffer composition of the present invention contains 5-80 mM TMAC, the concentration of KCl may be 40-90 mM, preferably 50-80 mM, and the concentration of (NH ₄ ) ₂ SO ₄ may be 1-7 mM, It is preferably 1.5-6 mM.

Therefore, when TMAC is included, the optimal PCR buffer composition of the present invention may include 15 to 70 mM TMAC; 50 to 80 mM KCl; 1.5 to 6 mM (NH ₄ ) ₂ SO ₄ ; and a final pH of 8.0 to 9.0.

The PCR buffer composition of the present invention may further include Tris. Cl and MgCl ₂ may further include Tween 20 and bovine serum albumin (BSA).

The invention provides a PCR kit for detecting a gene mutation or a SNP comprising the PCR buffer composition.

The PCR kit of the present invention may further include a DNA polymerase of Taq polymerase consisting of the amino acid sequence of SEQ ID NO: 1, and the DNA polymerase may include (a) the amino acid sequence of SEQ ID NO: 1 The substitution of the 507th amino acid residue in the amino acid sequence in (b) the 536th amino acid residue, the 660th amino acid residue in the amino acid sequence of SEQ ID NO: 1 or The 536th and 660th are two amino acid residue substitutions.

According to another preferred embodiment of the present invention, the substitution of the 507th amino acid residue is glutamic acid (E) by lysine (K), and the substitution of the 536th amino acid residue The substitution is the substitution of arginine (R) with lysine (K), and the substitution of the 660th amino acid residue is the substitution of arginine (R) with valine (V).

The PCR kit of the present invention may include any reagents or other elements known to those skilled in the art during primer extension.

According to another preferred embodiment of the present invention, the PCR kit includes one or more matching primers, one or more mismatching primers, or one or more matching primers and one or more mismatching primers. The matching primer and one or more mismatched primers hybridize to the target sequence, and the mismatched primer contains a non-canonical nucleotide at the 3 'end to the 7th base position of the hybridized target sequence. .

The PCR kit of the present invention may further comprise: (i) nucleoside triphosphate; (ii) a quantitative reagent that binds to double-stranded DNA; (iii) a polymerase blocking antibody; (iv) one or more control values or Control sequence; (v) one or more templates.

The "Taq polymerase" is a thermostable DNA polymerase named after the thermophilic bacteria Thermus aquaticus , and was originally isolated from the bacteria. The aquatic thermobacteria are bacteria that inhabit hot springs and hot water jets. Taq polymerase is confirmed to be an enzyme that can withstand the conditions of protein denaturation (high temperature) required during PCR. The optimal activity temperature of Taq polymerase is 75-80 ℃, the half-life is more than 2 hours at 92.5 ℃, 40 minutes at 95 ℃, 9 minutes at 97.5 ℃, and within 10 seconds at 72 ℃ Can replicate 1000 base pairs of DNA. This lacks a 3 → '5' exonuclease correction activity, and the error rate is measured in about 1 out of 9,000 nucleotides. For example, when heat-resistant Taq is used, PCR can be performed at a high temperature (60 ° C or higher). For Taq polymerase, the amino acid sequence shown in SEQ ID NO: 1 was used as a reference sequence.

In the present invention, in the amino acid sequence of SEQ ID NO: 1, the Taq polymerase in which the glutamic acid (E) of the 507th amino acid residue is replaced with lysine (K) is named "E507K "(SEQ ID NO: 2, the base sequence is SEQ ID NO: 7); in the amino acid sequence of SEQ ID NO: 1, the glutamic acid (E) of the 507th amino acid residue is replaced with lysine Amino acid (K), Taq polymerase with arginine (R) at the 536th amino acid residue replaced with lysine (K) was named "E507K / R536K" (SEQ ID NO: 3, base Base sequence is SEQ ID NO: 8); in the amino acid sequence of SEQ ID NO: 1, the glutamic acid (E) of the 507th amino acid residue is replaced with the lysine (K), the 660th Taq polymerase in which the arginine residue (R) of the amino acid residue was replaced with valine (V) was named "E507K / R660V" (SEQ ID NO: 4, the base sequence is SEQ ID NO: 9) ; Finally, in the amino acid sequence of SEQ ID NO: 1, the glutamic acid (E) of the 507th amino acid residue was replaced with lysine (K), and the 536th amino acid residue was refined. The amino acid (R) was replaced with lysine (K), and the 660th amino acid residue of arginine (R) was replaced with valine (V). Taq polymerase was named "E507K / R536K / R660V "(SEQ ID NO: 5, base sequence is SEQ ID NO: 10).

According to an embodiment of the present invention, the DNA polymerase distinguishes between matching primers and mismatching primers, the matching primers and mismatching primers hybridize with the target sequence, and the mismatching primers, for the hybridization target sequence, are at 3 The 'terminus may contain atypical nucleotides.

The mismatched primers are hybrid oligonucleotides that must be sufficiently complementary to hybridize to the target sequence, but do not reflect the exact sequence of the target sequence.

The "canonical nucleotide" or "complementary nucleotide" refers to standard Watson-Crick base pairs, A-U, A-T, and G-C.

The "non-canonical nucleotide" or "non-complementary nucleotide" refers to AC, AG, GU, GT, TC, TU, A, GG, TT, UU, CC, CU.

According to a preferred embodiment of the present invention, the amplification of the target sequence including the matching primers can show a lower Ct value than the amplification of the target sequence including the mismatched primers. .

For example, by covalently linking one or more nucleotides to a primer, a DNA polymerase can extend a matched primer more efficiently than a primer mismatched in a target sequence-dependent manner. Here, the higher efficiency can be observed by the following example, as in RT-PCR, the Ct value of the matched primer is lower than that of the mismatched primer. The difference in Ct value between the matched primer and the mismatched primer is 10 or higher, preferably 10-20, or there is no amplicon synthesis caused by the mismatched primer.

For example, in the first reaction, matched forward and reverse primers are used. In the second reaction, with the same experimental setup, mismatched forward primers and matched reverse primers are used. The product formed by standard PCR. One response is larger than the second response.

The Ct (Threshold Cross Cycle) value represents the DNA quantification method for quantitative PCR, and depends on the number of fluorescence cycles in which the log phase is plotted. The threshold for DNA-based fluorescence detection is set at least slightly higher than the background. The number of cycles where the fluorescence exceeds a threshold is called Ct or Cq (quantification cycle) according to the MIQE criterion. The Ct value for a given reaction is defined as the number of cycles in which the fluorescence emission crosses a fixed limit. For example, SYBR Green I and fluorescent probes can be used for real-time PCR of template DNA quantification. Fluorescence from the sample was collected each cycle during the PCR and plotted against the number of cycles. The initial template concentration is inversely proportional to the initial display time of the fluorescent signal. The higher the template concentration, the earlier the signal appears (appears at lower cycle numbers).

The present invention also relates to a nucleic acid sequence encoding the aforementioned DNA polymerase, and a vector and a host cell comprising the same. Various vectors can be prepared using a nucleic acid encoding a DNA polymerase of the present invention. Any vector including replicon and control sequences derived from a species interchangeable with the host cell can be used. The vector of the present invention may be an expression vector and include a nucleic acid region operatively linked to a nucleic acid sequence encoding a DNA polymerase of the present invention to control transcription and translation. A regulatory sequence refers to a DNA sequence required to express an operably linked coding sequence in a particular host organism. For example, regulatory sequences suitable for use in prokaryotes include a promoter, any operating sequences, and an alc ribosome binding site. In addition, the vector may contain a "Positive Retroregulatory Element (PRE)" to enhance the half-life of the transcribed mRNA. Controlled transcription and translation nucleic acid regions are generally suitable for host cells used to express polymerases. Various types of suitable expression vectors and suitable regulatory sequences for various host cells are known in the art. Generally, transcription and translation control sequences may include, for example, promoter sequences, ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, and enhancer or promoter sequences. In typical embodiments, the regulatory sequences include promoters and transcription initiation and termination sequences. Vectors also typically contain a multi-bit point adaptor region containing several restriction sites for insertion of foreign DNA. In certain embodiments, a "fusion tag" is used to facilitate purification and, if necessary, a tag / tag is subsequently removed (eg, "His-tag"). However, these are usually unnecessary when purifying thermoactive and / or thermostable proteins from a mesophilic host (eg, E. coli) using a "heating step". Standard recombinant DNA techniques are used to prepare suitable vectors containing the coding DNA replication sequences, regulatory sequences, phenotypic selection genes, and mutant polymerases of interest. As known in the art, isolated plastids, viral vectors, and DNA fragments are broken down and cut and ligated together in a particular order to produce the desired vector.

In a preferred embodiment of the present invention, the expression vector contains a selection marker gene for selecting a transformed host cell. Selection genes are known in the art and will vary depending on the host cell used. Suitable selection genes may include genes encoding ampicillin and / or tetracycline resistance, whereby cells in which these vectors are cultured can be transformed in the presence of these antibiotics.

In a preferred embodiment of the present invention, a nucleic acid sequence encoding a DNA polymerase of the present invention is introduced into a cell alone or in combination with a vector. Introduction or equivalent expression means that the nucleic acid enters the cell in a manner suitable for subsequent integration, expansion, and / or expression. Introduction methods include, for example, CaPO ₄ precipitation, liposome fusion, LIPOFECTIN ^® , electrophoresis, virus infection, and the like.

Prokaryotes are used as host cells in the initial transplantation step of the present invention. This is particularly useful for the rapid preparation of large amounts of DNA, the preparation of single-stranded DNA templates for generating site-oriented mutations, the simultaneous screening of many mutants, and the sequencing of the resulting mutants. Suitable host cells for prokaryotic cells include E. coli E. coli K12 strain 94 (ATCC No. 31, 446), E. coli E. coli strain W3110 (ATCC No. 27, 325), and E. coli K12 strain DG116 ( ATCC No. 53,606), E. coli X. 1776 (ATCC No. 31, 537) and E. coli B; many different strains of E. coli such as HB101, JM101, NM522, NM538, NM539 , And other species, including Bacillus family such as Bacillus subtilis , other Enterobacteriaceae such as Salmonella typhimurium , Serratia marcesans , and various Pseudomonas A species ( Pseudomonas sp. ) Can be used as a host. Typically, plastids used to transform E. coli include pBR322, pUCI8, pUCI9, pUCI18, pUC 119, and Bluescript M13. However, many other suitable carriers can also be used.

The invention also provides a method for preparing a DNA polymerase, comprising the steps of: culturing the host cell; and isolating the DNA polymerase from the culture and its culture supernatant.

The DNA polymerase of the present invention is prepared by culturing a host cell transformed with an expression vector containing a nucleic acid sequence encoding a DNA polymerase under appropriate conditions that induce or cause expression of the DNA polymerase. Methods for culturing transformed host cells under conditions suitable for protein expression are known in the art. A suitable host cell for preparing a polymerase from a plastid vector containing a lambda pL promoter is E. coli strain DG116 (ATCC No. 53606). Depending on the expression, polymerase can be collected and isolated.

Once purified, the mismatch discrimination of the DNA polymerase of the invention can be determined. For example, the mismatch discriminating activity is measured by comparing the amplification of a target sequence in which the primer perfectly matches the amplification of a single base mismatched target sequence in the 3 'end of the primer. Amplification can be detected immediately, for example by using a TaqManTM probe. The ability of a polymerase to distinguish between two target sequences can be estimated by comparing the Ct of the two reactions.

The invention provides a detection method for detecting at least one genetic mutation or SNP in vitro from more than one template by using the PCR kit.

The term "SNP (Single Nucleotide Polymorphism, Single Nucleotide Polymorphisms)" refers to a genetic change or mutation that shows a difference in one nucleotide sequence (A, T, G, or C) in a DNA nucleotide sequence.

In the in vitro SNP detection method of the present invention, the target sequence may be present in the test sample and includes DNA, cDNA or RNA, and preferably includes genomic DNA. The test sample may be a cell lysate prepared from bacteria, bacterial culture, or cell culture. In addition, the test sample may be contained in an animal, preferably in a vertebrate, and more preferably in a human subject. The target sequence may be contained in genomic DNA, preferably in the genomic DNA of the individual, more preferably in bacteria or vertebrates, and most preferably in the genomic DNA of a human subject.

The SNP detection method of the present invention may include a melting temperature analysis using a double-strand specific dye such as SYBR Green I.

Melting temperature curve analysis can be performed in real-time PCR equipment such as ABI 5700/7000 (96-well format) or ABI 7900 (384-well format) including onboard software SDS 2.1. Alternatively, the melting temperature curve analysis may be performed as an end point analysis.

The "double-stranded DNA-binding dye" or "double-strand-specific dye" can be used in the case where the double-stranded DNA has high fluorescence when combined with the double-stranded DNA, as compared with the case where the double-stranded DNA is not bound. Examples of such dyes include SOYTO-9, SOYTO-13, SOYTO-16, SOYTO-60, SOYTO-64, SYTO-82, ethyl bromide (EtBr), SYTOX Orange, TO-PRO-1, SYBR Green I, TO-PRO-3 or EvaGreen. In addition to EtBr and EvaGreen (Quiagen), these dyes have been used for immediate application testing.

The in vitro SNP detection method of the present invention can be analyzed by real-time PCR, agarose gel analysis after standard PCR, gene mutation-specific amplification or allele-specific amplification, and four-primer amplification by real-time PCR. The mutation system is performed by PCR or isothermal amplification.

The so-called "standard PCR" is a technique known to a person skilled in the art to amplify single or multiple copies of DNA or cDNA. Almost all PCRs use Taq polymerase or a thermostable DNA polymerase such as Klen Taq. A DNA polymerase enzymatically assembles a new DNA strand from a nucleotide by using a single strand of DNA as a template and using an oligonucleotide (primer). Amplicons generated by PCR can be analyzed on an agarose gel.

The "instant PCR" can monitor the process in real time while performing PCR. Therefore, information is collected throughout the PCR process, not at the end of the PCR. In real-time PCR, the characteristic of the reaction is the time point at which amplification is first detected in the cycle, rather than the target amount accumulated after a fixed number of cycles. Quantitative PCR is mainly performed using two methods, dye-based detection and probe-based detection.

The "Allele Specific Amplification (ASA)" is an amplification technique in which PCR primers are designed as a single nucleotide residue to distinguish other templates.

The "gene mutation-specific amplification or allele-specific amplification by instant PCR" can detect a gene mutation or SNP in an efficient manner. Unlike most other methods for detecting genetic mutations or SNPs, pre-amplification of the target genetic material is not necessary. ASA combines amplification and detection in a single reaction based on the difference between matched and mismatched primer / target sequence complexes. The increase in amplified DNA during the reaction can be monitored immediately by an increase in the fluorescent signal caused by a dye such as SYBR Green I, which emits light when combined with double-stranded DNA. Through real-time PCR of genetic mutation-specific amplification or allele-specific amplification, a fluorescent signal is delayed or the signal does not exist when a mismatch occurs. In a genetic mutation or SNP test, this provides information about the presence of a genetic mutation or SNP.

The "four-primer amplification hindered mutation system PCR" amplifies wild-type and mutant alleles together with control fragments in a single-tube PCR reaction. Non-allele-specific control amplicons are amplified by two universal (external) primers flanking the mutation region. The two allele-specific (internal) primers are designed in the opposite direction to the universal primers, and together with the universal primers, wild type and mutant amplicons are amplified. As a result, the two allele-specific amplicons can be easily separated by standard gel electrophoresis because the positions of the mutations are asymmetric with respect to the universal (external) primers, and therefore have different lengths. The control amplicons provide internal controls for false negatives and failed amplifications, and at least one of the two allele-specific amplicons is always present in the four-primer amplification hindered mutation system PCR.

The "isothermal amplification" means that the nucleic acid amplification is performed at a lower temperature, and does not depend on a thermal cycler, and a preferred temperature does not need to be changed during the amplification. The temperature used in isothermal amplification can be between room temperature (22-24 ° C) and about 65 ° C, or at room temperature of about 60-65 ° C, 45-50 ° C, 37 ° -42 ° C, or 22-24 ° C. . Isothermal amplification products can be analyzed by gel electrophoresis, ELISA, ELOSA (Enzyme linked oligosorbent assay), real-time PCR, ECL (improved chemiluminescence), analysis of RNA, DNA and proteins or turbidity. Wafer-based capillary electrophoresis device bioanalyzer for detection.

In one embodiment of the present invention, the E507K / R536K, E507K / R660V, or E507K / R536K / R660V Taq polymerase is used to confirm whether the mismatch primer extension ability of the template containing SNP (rs1408799, rs1015362, and / or rs4911414) cut back.

As a result, as shown in FIGS. 6 to 8, compared to the E507K Taq polymerase, it was confirmed that the amplification delay of the E507K / R536K, E507K / R660V or E507K / R536K / R660V Taq polymerase caused by mismatched primers, and its effect It is most obvious in E507K / R536K / R660V Taq polymerase.

This confirms that the DNA polymerase of the present invention has a higher selectivity for mismatch extension compared to the conventional Taq polymerase (E507K). Therefore, it is expected that the DNA polymerase of the present invention can be effectively applied to medical diagnosis of diseases and research of recombinant DNA.

According to another preferred embodiment of the present invention, the PCR kit includes one or more matching primers, one or more mismatching primers, or one or more matching primers and one or more mismatching primers. The matching primer and one or more mismatched primers hybridize to the target sequence, and the mismatched primer contains a non-canonical nucleotide at the 3 'end to the 7th base position of the hybrid target sequence .

The PCR kit of the present invention may further include a nucleoside triphosphate.

The PCR kit of the present invention also includes (i) one or more buffers; (ii) a quantitative reagent that binds to double-stranded DNA; (iii) a polymerase blocking antibody; (iv) one or more control values or control sequences (V) one or more templates; as shown.

Hereinafter, the present invention will be described in detail through examples. These embodiments are exemplary illustrations of the present invention, and those with ordinary knowledge in the art should understand that the scope of patent application of the present invention is not limited to the embodiments.

Example 1 Inducing Taq Polymerase Mutation

1-1. Fragment PCR

In this example, a lysine-substituted Taq DNA polymerase (hereinafter referred to as "R536K") of the 536th amino acid residue in the amino acid sequence of SEQ ID NO: 1 was prepared according to the following method: ), The 660th amino acid residue of arginine is replaced by valine acid Taq DNA polymerase (hereinafter referred to as "R660V"), and the 536th amino acid residue of arginine is lysine Taq DNA polymerase (hereinafter referred to as "R536K / R660V") in which the arginine residue of the 660th amino acid residue is substituted with valine.

First, Taq DNA polymerase fragments (F1 to F5) were amplified by PCR using the mutation-specific primers shown in Table 1, as shown in FIG. 1 (a). The reaction conditions are shown in Table 2.

The PCR product was confirmed by electrophoresis. As a result, as shown in FIG. 1 (b), the band of each fragment was confirmed, thereby confirming that the target fragment had been amplified.

1-2. Overlap PCR

Each fragment amplified in the 1-1 was used as a template, and primers (Eco-F and Xba-R primers) were used at both ends to amplify the full length. The reaction conditions are shown in Tables 3 and 4.

As a result, as shown in FIG. 1 (c), it was confirmed that "R536K", "R660V", and "R536K / R660V" Tag polymerase were amplified.

1-3. Connection

Under the conditions shown in Table 5, pUC19 was decomposed with the restriction enzyme EcoRI / XbaI at 37 ° C for 4 hours and the DNA was purified. Under the conditions shown in Table 6, the purified DNA was treated with SAP at 37 ° C for 1 hour. To prepare a carrier.

For inserts, the overlapping PCR products of Examples 1-2 were purified and decomposed with the restriction enzyme EcoRI / XbaI at 37 ° C for 3 hours under the conditions shown in Table 7. Gel extraction was performed together (Figure 2).

Under the conditions shown in Table 8, after connecting at room temperature (RT) for 2 hours, E. coli E. coli DH5α was transformed and screened on a medium containing ampicillin. The plastids prepared from the obtained colonies were sequenced to obtain Taq DNA polymerase mutants ("R536K", "R660V", and "R536K / R660V") having introduced the desired mutations.

Example 2 Introduction of E507K mutation

2-1. Fragment PCR

The activities of the "R536K", "R660V", and "R536K / R660V" Taq DNA polymerases prepared in Example 1 were tested, and the results confirmed that the activity was decreased (data not shown). "R536K" and "R660V" And "R536K / R660V" introduced the E507K mutation (in the amino acid sequence of SEQ ID NO: 1, the glutamic acid at the 507th amino acid residue was replaced with lysine). As a control group, wild-type (WT E507K mutation was also introduced into Taq DNA polymerase. The preparation method of Taq DNA polymerase introduced with E507K mutation is the same as that of Example 1.

Taq DNA polymerase fragments (F6 to F7) were amplified by PCR using the mutation-specific primers shown in Table 9, as shown in FIG. 3. The reaction conditions are shown in Table 10.

2-2. Overlap PCR

Each fragment amplified in the 2-1 was used as a template, and primers (Eco-F and Xba-R primers) were used at both ends to amplify the full length. The reaction conditions are shown in Table 11.

2-3. Connection

Under the conditions shown in Table 5, pUC19 was decomposed with the restriction enzyme EcoRI / XbaI at 37 ° C for 4 hours and the DNA was purified. Under the conditions shown in Table 6, the purified DNA was treated with SAP at 37 ° C for 1 hour. To prepare a carrier.

For the insert, the overlapping PCR product of Example 2-2 was purified, and was decomposed with the restriction enzyme EcoRI / XbaI at 37 ° C for 3 hours under the conditions shown in Table 7, and then was combined with the prepared vector. Gel extraction was performed together (Figure 4).

Under the conditions shown in Table 8, after connecting at room temperature (RT) for 2 hours, E. coli E. coli DH5α or DH10β was transformed and screened on a medium containing ampicillin. The plastids prepared from the obtained colonies were sequenced to obtain Taq DNA polymerase mutants ("E507K / R536K", "E507K / R660V", and "E507K / R536K / R660V") to which a desired mutation was introduced.

Example 3 qPCR using the DNA polymerase of the present invention

Using Taq polymerases each containing the "E507K / R536K", "E507K / R660V", and "E507K / R536K / R660V" mutations obtained in Example 2 was used to confirm whether the ability to extend mismatched primers was reduced for SNP-containing templates . As a control group, "E507K" Taq polymerase containing an E507K mutation was used.

The SNP-containing templates used in the present invention are rs1408799, rs1015362, and rs4911414. The genotype of each template and the sequence information of its specific primers (IDT, USA) are shown in Tables 12 and 13.

The PCR conditions (Applied Biosystems 7500 Fast) are shown in Table 14 below.

The probes are double labeled as shown in Table 15 below.

Oral epithelial cells were collected using an oral epithelial cell collection kit purchased from Noble Bio, dissolved in 500 μl of a lysis solution, and centrifuged at 12,000 × g for 3 minutes. The supernatant was transferred to a new tube using 1 μl each time (Figure 5).

Table 16 shows the reaction conditions and Table 17 shows the composition of the reaction buffer.

Except for the specific primers shown in Table 13, other reaction solutions were prepared in two test tubes in the same manner, and qPCR was performed by adding each allele-specific primer. At this time, the fluorescence signals detected in each test tube were combined to calculate and analyze the difference in the cycle (Ct) value of the fluorescence value reaching the threshold on the AB 7500 software (v 2.0.6). The longer the delay of the Ct value in the amplification caused by the mismatched primer, the better the mutation specificity or the allele specificity.

Results of AS-qPCR performed on rs1408799, rs1015362, and rs4911414, as shown in Figures 6 to 8, compared to the control group E507K, can confirm the Taq polymerization containing E507K / R536K, E507K / R660V or E507K / R536K / R660V mutation In the case of enzymes, the amplification delay caused by mismatched primers has the most obvious effect in E507K / R536K / R660V mutations.

This confirms that the Taq DNA polymerase of the present invention containing E507K / R536K, E507K / R660V or E507K / R536K / R660V mutations has more excellent mismatch extension selectivity than the Taq polymerases containing E507K mutation. Therefore, it is expected that the Taq DNA polymerase of the present invention can be effectively applied to medical diagnosis of diseases and research on recombinant DNA.

Example 4 Optimization of KCl concentration in reaction buffer

In this example, in order to find a high cation concentration that maximizes the delay in amplification caused by mismatch without reducing the amplification efficiency caused by matching, the KCl concentration in the PCR reaction buffer is adjusted to determine the optimal KCl concentration.

Taq polymerases containing the "E507K / R536K", "E507K / R660V", or "E507K / R536K / R660V" mutations obtained in Example 2 were used, respectively, and the KCl concentration thresholds were compared with Taq polymerases including the E507K mutation.

The template containing the SNP was rs1408799, the template genotype was TT, and the primers were the rs1408799 primers listed in Table 12. The qPCR conditions (Applied Biosystems 7500 Fast) were performed according to the conditions shown in Table 14. The double-labeled probes used 1408799-FAM of Table 15. The reaction conditions are shown in Table 18. The reaction buffer composition is shown in Table 19.

The results are shown in Figure 9. The KCI concentration threshold of E507K / R536K / R660V Taq polymerase is the lowest, and the KCl concentration threshold of E507K / R536K and E507K / R660V is lower than that of E507K.

Based on the above results, in order to determine the appropriate KCl concentration, further experiments were performed using E507K / R536K / R660V Taq polymerase. The primers used were rs1408799-T specific primers described in Table 13. qPCR conditions (Applied Biosystems 7500 Fast) were performed 35 cycles under the conditions of Table 14, and the reaction conditions are shown in Table 20.

The composition of the reaction buffer in the control group was the same as in Table 21. The composition of the reaction buffer in the experimental group was shown in Table 8. The concentration of (NH ₄ ) ₂ SO _{4 was} fixed at 2.5 mM, and the KCl concentration was changed in various ways.

The results of the PCR products were confirmed by electrophoresis after amplification under the conditions described in FIG. 10, and it was confirmed that it was possible to maximize the delay caused by mismatching without reducing the amplification efficiency caused by matching. A suitable KCl concentration is 75 mM.

Example 5 (NH ₄ ) ₂ SO ₄ Concentration optimization

This example is based on the results of Example 4, the KCl concentration in the reaction buffer is fixed at 75 mM, and the (NH ₄ ) ₂ SO ₄ concentration is changed in various ways to confirm the optimal (NH ₄ ) ₂ SO ₄ concentration. The primers used rs1408799-T specific primers described in Table 13. The qPCR conditions (Applied Biosystems 7500 Fast) were used for 35 cycles under the conditions of Table 14. The reaction conditions are shown in Table 20. The composition of the control buffer was the same as that in Table 21. the same.

As a result, as shown in FIG. 11, an appropriate (NH ₄ ) ₂ SO ₄ concentration was 5 mM.

Based on the results, the KCl concentration in the reaction buffer was fixed at 75 mM, and the concentration of (NH ₄ ) ₂ SO ₄ was set to about 5 mM (2.5 mM, 5 mM, and 10 mM, respectively). Amplification delay effect.

The primers used were rs1408799 primers described in Table 13, and the double-labeled probes were used 1408799-FAM in Table 15. The reaction conditions are shown in Table 22.

As a result, as shown in FIG. 12, the concentration of (NH ₄ ) ₂ SO _{4 had} the largest difference in Ct value at 10 mM. However, it was confirmed that the amplified Ct caused by the matching was slightly delayed and the peak was dumped downward. Therefore, the appropriate (NH ₄ ) ₂ SO ₄ was 5 mM.

The results of Example 4 and Example 5 were combined to confirm that the optimal reaction buffer combination contained 50 mM Tris. Cl, 2.5 mM MgCl ₂ , 75 mM KCl, 5 mM (NH ₄ ) ₂ SO ₄ , 0.1% Tween 20, and 0.01% BSA.

Example 6 Addition of TMAC to the reaction buffer and optimization of its concentration

In this example, TMAC was added to the reaction buffer, and its optimal concentration was confirmed. Based on the results of Examples 4 and 5, the concentration of KCl in the reaction buffer was fixed at 75 mM, and the concentration of (NH ₄ ) ₂ SO _{4 was} fixed at 5 mM. Various changes were made to the concentration of TMAC to confirm the most Good TMAC concentration.

E507K / R536K or E507K / R536K / R660V Taq polymerase was used. The template containing SNP was rs1408799, the template genotype was TT, and the primers were the rs1408799 primers listed in Table 12. The qPCR conditions (Applied Biosystems 7500 Fast) were performed under the conditions shown in Table 14. 1408799-FAM of Table 15 was used as the dual-labeled probe.

The experimental results are shown in FIG. 13. The appropriate TMAC concentration was confirmed to be 60 mM for E507K / R536K Taq polymerase and 25 mM for E507K / R536K / R660V Taq polymerase, and it was confirmed that if the TMAC concentration was too high, the amplification efficiency would decrease.

Example 7 KCl, (NH ₄ ) ₂ SO ₄ And TMAC concentration optimization

In this example, based on the results confirmed in Example 6, E507K / R536K / R660V Taq polymerase was used to confirm the optimal KCl, (NH ₄ ) ₂ SO ₄ and TMAC concentrations in the reaction buffer.

Specifically, the TMAC concentration in the reaction buffer was fixed at 25 mM, the (NH ₄ ) ₂ SO ₄ concentration was fixed at 2.5 mM, and the KCl concentration was adjusted to 20, 40, 60, and 80 mM, respectively. Experiments were performed on two SNPs, rs1015362 and rs4911414. The template genotype was as described in Table 12. The primers were the rs1015362 and rs4911414 primers described in Table 13. The qPCR conditions (Applied Biosystems 7500 Fast) were performed under the conditions of Table 14. The probe used 1408799-FAM in Table 15, and the reaction conditions are shown in Table 22.

As a result of the experiment, as shown in FIG. 14, for two SNPs, when the appropriate KCl concentration was 60 mM and the concentration was 80 mM, a decrease in amplification efficiency was observed.

From the above results, it was confirmed that the optimal KCl concentration in the reaction buffer was 60 mM, the (NH ₄ ) ₂ SO ₄ concentration was 2.5 mM, and the TMAC concentration was 25 mM. More specifically, it was confirmed that for E507K / R536K polymerase, the use of 75mM KCl, 5mM (NH ₄ ) ₂ SO ₄ , and 60mM TMAC was the most effective; for E507K / R536K / R660V polymerase, the use of 60mM KCl, 2.5 mM (NH ₄ ) ₂ SO ₄ concentration, and 25 mM TMAC were the most effective.

<110> Gene Kester Limited

<120> PCR buffer composition for enhancing DNA polymerase activity with increased specificity of gene mutation

<130> 107E0252

<150> KR 10-2017-0088376

<151> 2017-07-12

<160> 30

<170> PatentIn version 3.5

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<400> 3

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<211> 832

<212> PRT

<213> Artificial sequence

<400> 4

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<211> 832

<212> PRT

<213> Artificial sequence

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<211> 2499

<212> DNA

<213> Artificial sequence

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<211> 2499

<212> DNA

<213> Artificial sequence

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<211> 2499

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<213> Artificial sequence

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<211> 2499

<212> DNA

<213> Artificial sequence

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<211> 2499

<212> DNA

<213> Artificial sequence

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<212> DNA

<213> Artificial sequence

<400> 11

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<211> 27

<212> DNA

<213> Artificial sequence

<400> 12

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<211> 27

<212> DNA

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<212> DNA

<213> Artificial sequence

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<212> DNA

<213> Artificial sequence

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<212> DNA

<213> Artificial sequence

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<212> DNA

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<212> DNA

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<212> DNA

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<212> DNA

<213> Artificial sequence

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<212> DNA

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<212> DNA

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<212> DNA

<213> Artificial sequence

<400> 23

<210> 24

<211> 20

<212> DNA

<213> Artificial sequence

<400> 24

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<212> DNA

<213> Artificial sequence

<400> 25

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<212> DNA

<213> Artificial sequence

<400> 26

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<212> DNA

<213> Artificial sequence

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<212> DNA

<213> Artificial sequence

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<212> DNA

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Claims (16)

A PCR buffer composition for enhancing DNA polymerase activity with increased specificity for gene mutations, wherein the composition comprises 25 to 100 mM KCl; 1 to 15 mM (NH ₄ ) ₂ SO ₄ ; and a final pH of 8.0 to 9.0 .     The PCR buffer composition for enhancing DNA polymerase activity enhancement with increased specificity of gene mutation as described in item 1 of the scope of patent application, wherein the KCl concentration is 60 to 90 mM.     The PCR buffer composition for enhancing DNA polymerase activity enhancement with increased gene mutation specificity as described in item 1 of the patent application range, wherein the (NH ₄ ) ₂ SO ₄ concentration is 2.5 to 8 mM. The PCR buffer composition for enhancing DNA polymerase activity enhancement with increased specificity of gene mutation as described in item 1 of the scope of patent application, wherein the KCl concentration is 70 to 80 mM, and the (NH ₄ ) ₂ SO ₄ concentration is 4 to 6 mM.     The PCR buffer composition for enhancing the activity of a DNA polymerase having increased specificity of a mutation of a gene as described in item 1 of the scope of patent application, further comprising 5 to 80 mM of tetramethylammonium chloride (TMAC).         The PCR buffer composition for enhancing DNA polymerase activity enhancement with increased specificity of gene mutation as described in item 5 of the scope of patent application, wherein the KCl concentration is 40 to 90 mM.     The PCR buffer composition for enhancing DNA polymerase activity enhancement with increased specificity of gene mutation as described in item 5 of the scope of the patent application, wherein the (NH ₄ ) ₂ SO ₄ concentration is 1 to 7 mM. The PCR buffer composition for enhancing DNA polymerase activity enhancement with increased specificity of gene mutation as described in item 5 of the scope of patent application, wherein the TMAC concentration is 15 to 70 mM, the KCl concentration is 50 to 80 mM, and The NH ₄ ) ₂ SO ₄ concentration is 1.5 to 6 mM. The PCR buffer composition for enhancing DNA polymerase activity enhancement with increased specificity of gene mutation as described in the first patent application scope, wherein the PCR buffer composition further comprises Tris. Cl and MgCl ₂ .     A PCR kit for detecting a gene mutation or a SNP, comprising the PCR buffer composition described in any one of claims 1 to 9 of the scope of patent application.         The PCR kit for detecting a gene mutation or SNP as described in item 10 of the patent application scope, wherein the PCR kit further comprises a DNA polymerase of Taq polymerase consisting of the amino acid sequence of SEQ ID NO: 1, The DNA polymerase comprises (a) the substitution of the 507th amino acid residue in the amino acid sequence of SEQ ID NO: 1; and (b) the 536th amine of the amino acid sequence of SEQ ID NO: 1 Substitution of amino acid residues, substitution of the 660th amino acid residue, or substitution of the 536th and 660th two amino acid residues.         The PCR kit for detecting a gene mutation or SNP as described in item 11 of the scope of the patent application, wherein the substitution of the 507th amino acid residue is glutamic acid (E) by lysine (K), The 536th amino acid residue is substituted by arginine (R) by lysine (K), and the 660th amino acid residue is substituted by arginine (R) by valine ( V) Replace.         The PCR kit for detecting a gene mutation or SNP as described in item 10 of the patent application scope, wherein the PCR kit further comprises: (i) a nucleoside triphosphate; (ii) a quantitative reagent for binding to double-stranded DNA; (iii) a polymerase blocking antibody; (iv) one or more control values or sequences; and (v) one or more templates.         A detection method for in vitro detection of at least one genetic mutation or SNP from more than one template using the PCR kit described in item 10 of the patent application scope.         The method of claim 14, wherein the PCR kit further comprises a Taq polymerase DNA polymerase consisting of the amino acid sequence of SEQ ID NO: 1, the DNA polymerase comprising (a) SEQ ID Substitution of the 507th amino acid residue in the amino acid sequence of NO: 1; and (b) the substitution of the 536th amino acid residue in the amino acid sequence of SEQ ID NO: 1, the 660th Substitution of amino acid residues or 536th and 660th substitutions of two amino acid residues.         The method according to item 15 of the scope of patent application, wherein the substitution of the 507th amino acid residue is glutamic acid (E) by lysine (K), and the substitution of the 536th amino acid residue is The substitution is the substitution of arginine (R) with lysine (K), and the substitution of the 660th amino acid residue is the substitution of arginine (R) with valine (V).